The animal cell genome has a bimodal pattern of DNA methylation with CpG islands upstream to housekeeping genes being unmodified while tissue specific genes are generally methylated, but then undergo targeted demethylation in their cell type of expression. Since methyl moieties serve to inhibit basal transcription, this profile serves as a global mechanism of gene regulation. In order to understand how this basic pattern is generated, transfection into embryonic and somatic cells will be used as a model system for identifying the cis acting signals involved in both de novo methylation and demethylation. These results will then be confirmed in transgenic mice. The biochemical mechanisms for demethylation has yet to be deciphered. Using a newly developed novel in vitro assay, however, it should now be possible to learn how this reaction works and then clone the factors needed for demodification. These studies will clearly pave the way for learning what controls methylation in vivo and should provide the know how for altering these patterns and thereby targeting gene expression changes in vivo. While still preliminary, this technology could prove useful for developing strategies for the treatment of some genetic diseases. Genomic imprinting is an important form of gene regulation which evidently plays a role in embryonic growth control and is also involved in several human diseases, such as the Prader Willi/Angelman syndrome and in the generation of certain tumors. DNA methylation is probably required for distinguishing between the maternal and paternal copies of imprinted genes and in this way maintain allele specific transcription. The mechanism of this process may be similar to that which regulates active and inactive X chromosome gene expression, but imprinting clearly utilizes novel control elements. Selected methyl loci in each imprinted domain may provide the gamete derived signal which marks each parental allele. By studying the methylation pattern of these genes after injection into fertilized oocytes it should be possible to decipher both the cis and trans acting factors which regulate the imprinting process.